A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression.

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.

Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation.

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

Development of a robust crystallization platform for immune receptor TREM2 using a crystallization chaperone strategy.

TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.

Induction of a Senescence-Like Phenotype in Cultured Human Fetal Microglia During HIV-1 Infection.

HIV-1 causes premature aging in chronically infected patients. Despite effective anti-retroviral therapy, around 50% of patients suffer HIV-associated neurocognitive disorders (HAND), which likely potentiate aging-associated neurocognitive decline. Microglia support productive HIV-1 infection in the brain. Elevated markers of cellular senescence, including p53 and p21, have been detected in brain tissues from patients with HAND, but the potential for microglia senescence during HIV-1 infection has not been investigated. We hypothesized that HIV-1 can induce senescence in microglia. Primary human fetal microglia were exposed to single-round infectious HIV-1 pseudotypes or controls, and examined for markers of senescence. Post-infection, microglia had significantly elevated: senescence-associated ß-galactosidase activity, p21 levels, and production of cytokines such as IL-6 and IL-8, potentially indicative of a senescence-associated secretory phenotype. We also found increased detection of p53-binding protein foci in microglia nuclei post-infection. Additionally, we examined mitochondrial reactive oxygen species (ROS) and respiration, and found significantly increased mitochondrial ROS levels and decreased ATP-linked respiration during HIV-1 infection. Supernatant transfer from infected cultures to naïve microglia resulted in elevated p21 and caveolin-1 levels, and IL-8 production. Finally, nucleoside treatment reduced senescence markers induction in microglia. Overall, HIV-1 induces a senescence-like phenotype in human microglia, which could play a role in HAND.

Fate of microglia during HIV-1 infection: From activation to senescence?

Microglia support productive human immunodeficiency virus type 1 (HIV-1) infection and disturbed microglial function could contribute to the development of HIV-associated neurocognitive disorders (HAND). Better understanding of how HIV-1 infection and viral protein exposure modulate microglial function during the course of infection could lead to the identification of novel therapeutic targets for both the eradication of HIV-1 reservoir and treatment of neurocognitive deficits. This review first describes microglial origins and function in the normal central nervous system (CNS), and the changes that occur during aging. We then critically discuss how HIV-1 infection and exposure to viral proteins such as Tat and gp120 affect various aspects of microglial homeostasis including activation, cellular metabolism and cell cycle regulation, through pathways implicated in cellular stress responses including p38 mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB). We thus propose that the functions of human microglia evolve during both healthy and pathological aging. Aging-associated dysfunction of microglia comprises phenotypes resembling cellular senescence, which could contribute to cognitive impairments observed in various neurodegenerative diseases. In addition, microglia seems to develop characteristics that could be related to cellular senescence post-HIV-1 infection and after exposure to HIV-1 viral proteins. However, despite its potential role as a component of HAND and likely other neurocognitive disorders, microglia senescence has not been well characterized and should be the focus of future studies, which could have high translational relevance. GLIA 2017;65:431-446.